PROJECT SUMMARY The branched architecture of the airways of the lungs permit the transfer of approximately six liters of air per minute between the external surroundings and the alveoli. The airway epithelial tree accomplishes gas exchange, mucus production, and pathogen clearance and blocks the entry of water, particulates, and microbes. To accomplish these diverse biological functions, the airway epithelium is comprised of several distinct cell types that differentiate from common progenitors during embryonic development, the first of which is the pulmonary neuroendocrine cell. Disrupting the differentiation of the specialized epithelial cell types negatively affects airway morphogenesis, and abnormally high numbers of pulmonary neuroendocrine cells are found in several congenital and acquired diseases of the lung. As it differentiates, the epithelium secretes ions and water across its apical surface, causing fluid to fill the lumen of the airways with a transmural pressure high enough to inflate the lungs. Defects that cause a decrease in transmural pressure are associated with both underdeveloped lungs and an increase in pulmonary neuroendocrine cells, but the specific role of pressure and the molecular signaling downstream of this mechanical cue are unknown. By combining time- lapse confocal imaging with an innovative microfluidic culture system, we found that transmural pressure controls the rate of lung development and the expression of markers of neuroendocrine cells. Using next- generation sequencing analysis, we found that low transmural pressure decreases the expression of targets of Notch, the master regulator of pulmonary neuroendocrine differentiation, and YAP, a known mechanosensor. Here, we hypothesize that transmural pressure coordinates the growth and differentiation of the different cell types within the epithelium by signaling through Notch and YAP. We will combine microfluidic devices with engineered mice, high-resolution time-lapse spinning disk confocal microscopy, and next-generation sequencing analysis to define the relative roles of pressure, Notch, and YAP in the regulation of pulmonary neuroendocrine progenitor fate decisions. In Specific Aim 1, we will use microfluidic chest cavities, engineered mice, time-lapse imaging, and single cell RNA-sequencing to define physically how transmural pressure regulates the pulmonary neuroendocrine population in the developing lung. In Specific Aim 2, we will use microfluidic chest cavities, reporter mice, and chromatin immunoprecipitation approaches to define whether and how transmural pressure regulates Notch signaling in the embryonic airway epithelium. In Specific Aim 3, we will determine whether pressure signals through YAP to affect pulmonary neuroendocrine differentiation and the Notch pathway. This work will define how mechanical signals from the microenvironment are transmitted to the first progenitor fate decision in the developing airway epithelium. We expect that our results will reveal novel insights into mechanical control of progenitor differentiation during tissue development and suggest new therapeutic targets for defects in lung development.